Reactive oxygen species (ROS) are produced in distinct cellular locations - by the organelle location of oxidases and mitochondria - and exert their effects only nanometers from the site of production. Little is known about how cells regulate production of reactive oxygen species to control signal transduction. The objective of this application is a detailed quantitative analysis of the local intercellular regulation of ROS ad its control over immunological processes. Observations of mitochondrial migration during T cell activation have been largely attributed to local ATP demands; however our findings of active redox signal regulation and quantitative modeling of intracellular H2O2 leads us to hypothesize that T cells coordinate mitochondrial movement to the immunological synapse (IS) to control T cell activation signaling via ROS production. The long-term rationale for this research is that by understanding how ROS is used to oxidize proteins during signaling, methods of targeting mitochondrial function can be appropriately applied to augment or suppress self- or antigenic peptide presentation. This project will yield new experimental platforms and analytical tools for cellular interaction studies in conjunction with quantitative insight of T cell responsiveness as a function of metabolic phenotype. First, we will develop a high-throughput acquisition platform for monitoring T cell/antigen-presenting cell engagement. Secondly, single cell frequency response signatures from oxidative stimuli will be related to T cell activation and IS features. Finally, we will model regional oxidation during TCR ligation to test the hypothesis that the mitochondrial movement that occurs during IS formation creates oxidized zones proximal to the pMHC:TCR interface. These technologies will be used to determine the contributions of localized ROS sources to T cell signaling and investigate spatiotemporal relationships between ROS generation and calcium. The proposed research is innovative because it merges the technological developments of new modeling methods and microfluidic platforms to address the challenge of analyzing local oxidation during T cell signaling. The outcomes of this work are expected to fundamentally advance our understanding of how cells use spatially distinct ROS sources to regulate receptor-initiated signaling. This knowledge will have large impact in ultimately redefining intracellular oxidation by more biologically relevant metrics for diagnosis and treatment of diseases.